Since B cell lymphoma in man is a clonal tumor, it is an ideal setting in which to study clonal diversity and clonal progression. The continuing goals of this project are to define two signal events that characterize the natural history of human lymphoma: 1. Clonal Evolution: the generation of clonal diversity; 2. Clonal Transformation: the conversion to biologically aggressive disease. Our preliminary data suggest that prior to therapeutic interventions, clonal diversity is a low frequency event that may vary with histologic subcategory and increase with time. Likewise, clonal transformation may arise from escape from immunoregulatory control mechanisms, the activation of oncogenes or the deletion of putative "antioncogenes". We have developed a cell fusion system that provides stable tumor heterohybrids that secrete large quantities of clonotypic lymphoma Ig. This has provided us with the ability to evaluate clonal diversity by preparing families of mouse monclonal antibodies directed against the idiotypic portions of the Ig molecule as well as to detect subtle Ig structural changes by isoelectric focusing in gels. When combined with Southern analysis of the DNA derived from these tumors and hybrids, this approach provides a powerful system for the study of clonal diversity and biologic transformation. As part of our strategy we are also developing new and unique clonotypic markers that will potentially have broad application to these studies. These approaches include: 1. An examination of V gene usage with Vh gene specific probes in order to detect biased V gene usage and cross-reacting hybridoma idiotypes; 2. Characterization of deletions and amplifications in lymphoma DNA by a unique 2D gel electrophoresis system to identify genetic components of clonal progression and variation. The data generated in these studies will provide an immunologic and molecular genetic basis for human lymphoma clonal evolution. The construction of such a model will have great potential impact on: 1. Immunodiagnosis and early screening; 2. The development of a set of criteria for predicting prognosis; 3. The design of more effective treatment strategies. The overall impact of these studies is to provide the basis for increasing the cure rate of these highly lethal tumors.